OK－432联合IL－2对C57BL／6小鼠Lewis肺癌的抑制作用及p21的表达

目的：研究OK－432与IL－2抑制近交系小鼠C57BL／6 Lweis肺癌（LLC）的生长及p21的表达。方法：以C57BL／6荷瘤LLC小鼠为模型，观察OK－432联合IL－2对肿瘤发生、生长、移的影响，体内对肿瘤细胞的作用及用抗鼠p21单克隆抗体进行SABC免疫组化技术半定量测定p21在Lewis肺癌原发灶中的表达。结果：二联用对肿瘤体积和重量的抑制作用明显增强（P＜0．01），增强抑瘤率（P＜0．05）；肺转称灶数目明显减少，电镜下显示肿瘤灶中出现许多凋亡细胞、瘤细胞核染色质浓集成块，在核膜内呈新月形或环形排列，核膜清楚，瘤细胞内质网扩张，线粒体肿胀，p21在联合用药组表达明显减少。结论：OK－432与IL－2有明显的协同抗肿瘤活性，其杀瘤过程主要为对瘤细胞的直接或间接攻击，部位原因可能阻抑ras癌基因的激活，诱导瘤细胞凋亡；提示OK－432联合IL－2可为临床治疗癌症患一种有效的生物治疗方法。     

新CpG寡聚脱氧核苷酸体内抗肿瘤与增强免疫活性研究

目的:探讨新设计CpG寡聚脱氧核苷酸(ODN)在小鼠体内的抗肿瘤作用及其对荷瘤小鼠免疫功能的影响.方法:建立C57BL/6小鼠腹腔、皮下荷黑素瘤肿瘤模型,腹腔注射CpG ODN,观察荷瘤鼠生存时间、肿瘤生长曲线,计算抑瘤率.用酶联免疫吸附试验(ELISA)检测小鼠血清中白细胞介素(IL)12和免疫球蛋白E(IgE)含量;3H-TDR掺入法检测脾脏B细胞、T细胞增殖活性;51Cr释放法检测NK细胞杀伤活性;中性红法检测腹腔巨噬细胞吞噬功能.结果:CpG10,CpG11能明显延长腹腔接种肿瘤小鼠的生存时间,与阳性对照CpG1826相比P＜0.01;二者平均抑瘤率(皮下荷瘤鼠)分别为(55.2±2.3)%与(40.7±1.7)%,显著高于CpG 1826 [抑瘤率(17.8±7.6) %](P＜0.05,P＜0.01).CpG10/CpG11可促使荷瘤小鼠血清IL-12含量显著升高( P＜0.01),IgE含量显著下降( P＜0.01);明显刺激B细胞、T细胞增殖能力以及NK细胞的杀伤活性( P＜0.01),对腹腔巨噬细胞的吞噬功能有显著提高( P＜0.01).CpG10免疫增强活性较CpG1826更强( P＜0.05).结论:CpG ODN能激活荷瘤鼠抗肿瘤免疫反应,从而抑制小鼠黑素瘤的生长,新结构寡核苷酸的CpG10呈现优于阳性对照的良好抗肿瘤免疫效应.     

OK-432联合IL-2对C_(57)BL/6小鼠Lewis肺癌的抑制作用及p21的表达

目的　研究OK 4 32与IL 2抑制近交系小鼠C57BL/ 6Lweis肺癌 (LLC)的生长及p2 1的表达。方法　以C57BL/ 6荷瘤LLC小鼠为模型 ,观察OK 4 32联合IL 2对肿瘤发生、生长、转移的影响 ,体内对肿瘤细胞的作用及用抗鼠p2 1单克隆抗体进行SABC免疫组化技术半定量测定p2 1在Lewis肺癌原发灶中的表达。结果　二者联用对肿瘤体积和重量的抑制作用明显增强(P <0 .0 1) ,增强抑瘤率 (P <0 .0 5 ) ,肺转称灶数目明显减少 ,电镜下显示肿瘤灶中出现许多凋亡细胞、瘤细胞核染色质浓集成块 ,在核膜内呈新月形或环形排列 ,核膜清楚 ,瘤细胞内质网扩张 ,线粒体肿胀 ,p2 1在联合用药组表达明显减少。结论　OK 4 32与IL 2有明显的协同抗肿瘤活性 ,其杀瘤过程主要为对瘤细胞的直接或间接攻击 ,部位原因可能阻抑ras癌基因的激活 ,诱导瘤细胞凋亡 ;提示OK 4 32联合IL 2可为临床治疗癌症患者一种有效的生物治疗方法     

pcEgr-IFNγ基因治疗联合放射治疗抗肿瘤作用的实验研究

目的：探讨pcEgr-IFNγ基因－放射联合治疗对修黑色素瘤B16小鼠的抗肿瘤作用及其免疫学机理。方法：肿瘤局部注射脂质体包裹的pcEgr-IFNγ重组质粒后36h,接受20GyX射线照射,观察放疗后不同时间肿瘤大小,放疗后3d瘤内IFNγ转录水平和15d部分免疫学指标。结果pcEgr-IFNγ基因－放射联合治疗后第3－15天,肿瘤生长速率明显低于单纯照射组,照第第9－15天明显低于单纯基因治疗组和空质料对照组;治疗后第3天,pcEgr-IFNγ基因－放射联合治疗组瘤内IFNγ表达水平明显高于单纯基因治疗组;脾脏NK毒胞毒活性、IL－2和IFNγ分泌活性均明显高于单纯放疗组和单纯基因治疗组。结论：pcEgr-IFNγ基因－放射联合治疗抗肿瘤作用明显优于单纯放疗和单纯基因治疗组,其机理可能与放疗诱导瘤内IFNγ基因表达增强,激活荷瘤机体抗肿瘤免疫功能有关。     

大鼠种植型肝癌电化学疗法疗效评价的实验研究

目的　评价电化学疗法 (EChT)对大鼠种植型肝癌的治疗效果。材料与方法　采用直接注射法制作大鼠肝癌模型。应用影像学、病理学及系列免疫指标 (IL 2、sIL 2R、IL 6、TNF α)检测 ,生存期比较等方法观察荷瘤大鼠EChT治疗前后肿瘤大小、坏死、细胞凋亡、外周血系列免疫指标表达水平、生存期长短的动态变化 ,并与同期对照组进行比较。结果　EChT治疗前 ,大鼠肝肿瘤平均体积为 10 0± 6mm3 ;EChT治疗后 1周 ,大鼠肝肿瘤平均体积为12 5± 10mm3 ,而荷瘤对照组肝肿瘤平均体积为 190± 11mm3 ;治疗组肿瘤生长率为 2 5 % ,而荷瘤对照组肝肿瘤生长率为 90 % (P <0 .0 5 )。治疗组病理光镜下肿瘤明显坏死 ,并可明显诱发细胞凋亡。荷瘤大鼠EChT治疗前IL 2水平呈低表达 ,sIL 2R、IL 6、TNF α则高表达 ,与正常大鼠比较差异显著 (P <0 .0 5 ) ;EChT治疗后 ,与同期荷瘤大鼠对照组比较 ,IL 2表达水平升高 ,sIL 2R、IL 6、TNF α表达水平下降 (P <0 .0 5 )。EChT治疗组大鼠生存期明显延长 (P <0 .0 5 )。结论　肿瘤负荷可导致大鼠细胞因子表达紊乱。EChT治疗可明显抑制肿瘤生长 ,促进肿瘤坏死 ,诱发肿瘤细胞凋亡 ,改善和纠正机体免疫紊乱 ,增强大鼠免疫力 ,延长大鼠生存期。     

猴头菌多糖促进同基因骨髓移植小鼠免疫功能重建

同基因骨髓移植55d后,受体小鼠免疫功能严重缺损,脾细胞产生IL—2的能力等细胞免疫功能明显低下。经腹腔连续注射猴头菌多糖15d后,小鼠脾细胞产生IL—2的能力,对ConA刺激的增殖反应和混合淋巴细胞反应均显著增强;抗羊红细胞抗体(IgM)分泌细胞数明显增加。实验结果提示:猴头菌多糖能明显促进同基因骨髓移植小鼠免疫功能的早期恢复。     

细胞因子对小鼠脾单个核细胞Th17分化和IL-17分泌的影响

目的 研究外源性肿瘤坏死因子β(TGF-13)、白细胞介素(IL)-6和IL-23对小鼠脾单个核细胞Th17分化和IL-17分泌的影响,并初步探讨其在器官移植免疫中的调节机制.方法 用佛波酯(PMA)和离子霉素(Ion)刺激小鼠脾单个核细胞4h,将细胞因子与脾单个核细胞共培养72h,根据加入细胞因子的不同,将培养细胞分...     

pEgr-ssEndostatin重组质粒瘤内辐射诱导表达及其抗肿瘤作用

目的 观察pEgr-ssEndostatin基因-放射治疗对移植黑色素瘤B16小鼠的抗肿瘤作用及其在瘤内辐射诱导表达。方法 肿瘤局部注射脂质体包裹的pEgr-ssEndostatin重组质粒后42h，接受20Gyx射线照射，观察放疗后不同时间肿瘤大小，检测放疗后第2天瘤内Endostatin转录水平。结果 pEgr-ssEndostatin基因-放射治疗荷瘤小鼠能够增强放疗疗效；单纯接种pEgr-ssEndostatin质粒组和接种pEgr-ssEndostatin质粒 20Gy照射组小鼠的肿瘤组织内有Endostatin mRNA表达，且pEgr-ssEndostatin质粒照射组的EndostatinmRNA水平高于单纯pEgr-ssEndostatin质粒组。结论 pEgr-Endostatin基因-放射治疗抗肿瘤作用明显优于单纯放疗和单纯基因治疗，肿瘤局部转染重组质粒后照射瘤内Endostatin表达增强。     

羊栖菜多糖对荷瘤小鼠免疫功能的影响

目的观察羊栖菜多糖对S-180肉瘤移植小鼠免疫功能的影响，评价其抗肿瘤作用的机制。方法采用水煮醇沉、Sevag法并结合胰酶消化去蛋白及Sephadex G200柱分离纯化获得羊栖菜多糖SFP1和SFP2，用S-180肉瘤移植小鼠作为实验模型，用羊栖菜多糖SFP2腹腔注射给药，观察羊栖菜多糖对荷瘤小鼠免疫功能的调节作用。结果羊栖菜多糖能明显抑制S-180肉瘤在荷瘤小鼠上的生长，显著提高S-180荷瘤小鼠胸腺指数、脾指数及ConA诱导的脾淋巴细胞增殖，提高荷瘤小鼠NK细胞活性及腹腔巨噬细胞的吞噬活性。结论羊栖菜多糖能明显提高荷瘤小鼠机体的免疫功能，提高机体免疫功能是羊栖菜多糖抑制肿瘤生长的机制之一。     

低剂量辐射增强小鼠肿瘤基因-放射治疗效应的免疫学机制研究

目的 探讨低剂量辐射全身照射对小鼠Lewis肺癌移植肿瘤基因-放疗方案中的免疫增强作用机制。方法 小鼠右后肢皮下接种Lewis肺癌细胞建立荷瘤模型,基因-放疗组中小鼠肿瘤局部注射由多聚乙烯亚胺包裹的pEgr-IL18-B7.1重组质粒,分别接受由2 Gy 局部照射和0.075 Gy 全身照射组合的不同治疗方案,通过³H-TdR标记方法检测小鼠CTL和NK细胞毒活性,ELISA方法检测TNF-α和IFN-γ分泌活性,观察各治疗组对荷瘤小鼠抗肿瘤免疫的作用。结果 在pEgr-IL18-B7.1基因治疗方案中,单次大剂量辐射局部照射后加多次低剂量全身照射与常规多次大剂量辐射局部照射相比,小鼠CTL和NK细胞毒活性显著增强,TNF-α和IFN-γ分泌活性有不同程度的增高。 结论 低剂量辐射可以通过促进CTL和NK细胞毒效应,上调TNF-α和IFN-γ细胞因子表达,从而增强机体抗肿瘤免疫功能,提高肿瘤基因-放疗的抑瘤效果。     

Transarterial chemoembolization alone and in combination with other therapies: a comparative study in an animal HCC model

The purpose of this study is to compare transarterial chemoembolization (TACE) alone and in combination with other therapies in an animal model. Subcapsular implantation of a solid Morris hepatoma 3924A in the liver was carried out in 50 male ACI rats (day 0). Tumor volume (V1) was measured by MRI (day 13). After laparotomy and retrograde placement of a catheter into the gastroduodenal artery (day 14), the following protocols of the interventional procedure were applied: TACE (mitomycin C + lipiodol) + immunotherapy (group A: TNF + IL-2, group B: OK-432 + IL-2); TACE + antiangiogenesis therapy (group C: TNP-470, group D: endostatin); TACE alone in group E (control group). Tumor volume (V2) was assessed by MRI and the mean ratio of x (V2/V1) was calculated. Data were analyzed using Dunnetts t test (comparing therapeutic groups with the control group) and the Student-Newman-Keuls test (comparing significant therapeutic groups). Multivariate analysis showed a significant reduction in the tumor growth rate (P<0.05) in groups B (x=6.53) and C (x=4.01) compared to the mean ratio of the control group E (x=9.14). Significant results were observed in group C (P<0.05) in comparison with the other therapeutic groups. TACE combined with immunotherapy (OK-432) and antiangiogenesis therapy (TNP-470) retards tumor growth compared with TACE alone in an HCC animal model.The first two authors contributed equally to this publication.     

Intractable massive ascites following radical gastrectomy, treatment with local intraperitoneal administration of OK-432 using a unified CT and fluoroscopy system

The case of a patient who developed intractable massive ascites caused by hepatic lymphorrhea derived from surgical injury to the hepatoduodenal ligament is presented. OK-432 was injected intraperitoneally through a catheter advanced to near the hepatoduodenal ligament, so as to expose this site to a high concentration of OK-432. Under ultrasound guidance, it was difficult to reach this site due to massive ascites, and so we performed this procedure under CT and fluoroscopic guidance using a unified CT and angiography system. Subsequently, local administration of OK-432 on five separate occasions resulted in resolution of the ascites. We ascribe this favourable result to the use of this unified CT and angiography system to advance the catheter to the suitable site, making possible the local administration of OK-432.     

OK-432/IL-2对Lewis肺癌抑制作用的光镜及电镜观察

OK-432/IL-2治疗48只C_(57)BL/6小鼠体内移植性路易氏肺癌(Lewis lung cancer,LLC),光镜及电镜观察结果表明:Ok-432/IL-2能抑制LLC的生长和转移,并且有使癌细胞逆转的趋势。     

太空环境对黑色素瘤B16细胞成瘤性的影响

目的观察空间诱变小鼠黑色素瘤B16细胞株的成瘤性，寻找免疫原性发生变异的细胞株。方法将B16细胞株应用细胞低温长期生存系统，搭载于我国第20颗返回式卫星，返地后进行单克隆化，从得到的110株单克隆太空诱变B16细胞株中随机选取4株，常规培养后和对照细胞株同时分别接种C57BL／6J小鼠，腹腔接种组观察生存期，皮下接种组14d后取血、处死，记录瘤重，脾脏重，胸腺重，并进行血清细胞因子检测和瘤体的病理学检测。结果初步确定了适合太空诱变肿瘤细胞株体内筛选实验的方法和筛选指标，并筛选出了1株出瘤时间延迟、瘤重缩小，生存期延长，荷瘤小鼠血清IL-2浓度升高，免疫原性可能发生变异的B16细胞株。结论空间环境可使体外培养肿瘤细胞产生变异，能否通过太空诱变的方法增强肿瘤细胞株的免疫原性需进一步实验验证。     

Radiofrequency Ablation of Liver Tumors in Combination with Local OK-432 Injection Prolongs Survival and Suppresses Distant Tumor Growth in the Rabbit Model with Intra- and Extrahepatic VX2 Tumors

Purpose

To evaluate survival and distant tumor growth after radiofrequency ablation (RFA) and local OK-432 injection at a single tumor site in a rabbit model with intra- and extrahepatic VX2 tumors and to examine the effect of this combination therapy, which we termed immuno-radiofrequency ablation (immunoRFA), on systemic antitumor immunity in a rechallenge test.

Methods

Our institutional animal care committee approved all experiments. VX2 tumors were implanted to three sites: two in the liver and one in the left ear. Rabbits were randomized into four groups of seven to receive control, RFA alone, OK-432 alone, and immunoRFA treatments at a single liver tumor at 1 week after implantation. Untreated liver and ear tumor volumes were measured after the treatment. As the rechallenge test, tumors were reimplanted into the right ear of rabbits, which survived the 35 weeks and were followed up without additional treatment. Statistical significance was examined by log-rank test for survival and Student’s t test for tumor volume.

Results

Survival was significantly prolonged in the immunoRFA group compared to the other three groups (P < 0.05). Untreated liver and ear tumor sizes became significantly smaller after immunoRFA compared to controls (P < 0.05). In the rechallenge test, the reimplanted tumors regressed without further therapy compared to the ear tumors of the control group (P < 0.05).

Conclusion

ImmunoRFA led to improved survival and suppression of distant untreated tumor growth. Decreases in size of the distant untreated tumors and reimplanted tumors suggested that systemic antitumor immunity was enhanced by immunoRFA.     

Experimental intraperitoneal infusion of OK-432 in rats: Evaluation of peritoneal complications and pathology

Purpose

OK-432 is known to be a potent sclerosant of cystic lesions. The purpose of this study was to evaluate both its safety and pathologic effects after the infusion of OK-432 into the peritoneal cavity of rats.

Materials and methods

Twenty male rats were used in this study. Twelve rats were infused intraperitoneally with 0.2 Klinishe Einheit of OK-432 melted in 2 mL of normal saline (group 1: the treated group); four rats each were infused intraperitoneally with 0.5 mL of 99% ethanol (group 2) and normal saline (group 3), and served as the control groups. An abdominal ultrasonographic examination was performed both before and after the infusions in all rats. Three rats in group 1 and one rat in each of groups 2 and 3 were sacrificed each week following the infusion. Gross and microscopic evaluations of the peritoneum and abdominal cavity were performed on each rat.

Results

In group 1, the abdomen was clear on gross inspection and the peritoneum was unremarkable on microscopic examination. In group 2, mild-to-moderate peritoneal adhesions were revealed grossly, and inflammation and fibrosis of the peritoneum were demonstrated microscopically. In group 3, no specific abnormalities were noted on gross or microscopic examinations.

Conclusion

Leakage or abnormal infusion of OK-432 solution into the peritoneal cavity during sclerotherapy of intra-abdominal or retroperitoneal cystic lesions does not result in any significant complications.     

OK-432 sclerotherapy of plunging ranula in 21 patients: it can be a substitute for surgery

BACKGROUND AND PURPOSE: Although first-choice therapy for the ranula is surgery, this choice presents technical difficulties and frequent recurrences because of insufficient surgery. We evaluated the efficacy of OK-432 sclerosis of the plunging ranula as a substitute for surgery. METHODS: Twenty-one patients with plunging ranula were treated with intralesional injection of OK-432. The liquid content of the ranula was aspirated as much as possible, after which OK-432 solution was injected in the same volumes as that drawn out. Patients were followed on sonography or CT. RESULTS: Seven (33.3%) patients with plunging ranulas showed total shrinkage and resolution, and 4 (19%) patients showed near-total shrinkage (more than 90% of the volume). Four (19%) patients revealed marked shrinkage (more than 70% of the volume), and 3 (14.3%) patients showed partial shrinkage (less than 70% of the volume). Three (14.3%) patients showed recurrence after total shrinkage 1 month after injection. The overall recurrence rate after each injection was 47% (16 of 34 injections in 21 patients), but the recurrence rate after the last sclerotherapy was only 14%. There were no serious side effects except for fever lasting 2-3 days (12 patients) and swelling (10 patients) for 3-5 days. Mild odynophagia for 1-2 days was also noted in 7 patients, and there was 1 severe case of odynophagia. CONCLUSION: OK-432 sclerotherapy of plunging ranula is a safe and potentially curative procedure that may be used as a primary treatment for plunging ranula before considering surgery.     

Melanoma growth and tumorigenicity in models of microgravity

INTRODUCTION: Spaceflight involves numerous biological stressors that could affect long-term cancer incidence and tumor behavior. Ground-based models of microgravity can be used to investigate in vitro and in vivo tumor growth as a preparation for later work in space. The incidence of tumor growth and carcinogenesis in microgravity is as yet unknown. Hence, we investigated the effects of modeled microgravity on tumor growth and tumorigenicity using ground-based in vitro and in vivo models. METHODS: Murine B16-F10 melanoma cells were cultured in a tissue culture flask (FL) and in a rotating-wall vessel bioreactor (BIO) designed by NASA to simulate some aspects of microgravity. We then measured cell growth, melanin production, and apoptosis. After 48 h of cultures in FL and BIO, cells were inoculated subcutaneously in C57BL/6 mice, syngeneic hosts for B16-F10 tumor cells. Tumor sizes were then measured every other day. RESULTS: BIO cultures had 50% decreases in growth when compared with FL cultures while demonstrating an inversely proportional increase in doubling time. Melanin production (a marker of differentiation) increased at 24 and 48 h in BIO. Flow cytometry analysis demonstrated that there was an increase in the percentage of apoptotic cells in the BIO when compared with that in the FL. When BIO-cultured melanoma cells were inoculated subcutaneously in mice, there was a significant increase in tumorigenicity as compared with FL-cultured cells. CONCLUSION: Our results indicate that simulated microgravity may have altered the tumor cell characteristics and enhanced the invasive property. It is possible that the microgravity analogue culture environment may have selected highly tumorigenic cells for survival despite the decreased overall growth in the microgravity analogue.     

Small-animal PET of melanocortin 1 receptor expression using a 18F-labeled alpha-melanocyte-stimulating hormone analog.

(18)F-Labeled small synthetic peptides have emerged as attractive probes for imaging various molecular targets with PET. The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor [MC1R]) is overexpressed in most murine and human melanomas. It is a promising molecular target for diagnosis and therapy of melanomas. However, (18)F compounds have not been successfully developed for imaging the MC1R. METHODS: In this study, an alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH(2) (NAPamide), was radiolabeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The resulting radiopeptide was evaluated as a potential molecular probe for small-animal PET of melanoma and MC1R expression in melanoma xenografted mouse models. RESULTS: The binding affinity of (19)F-SFB-conjugated NAPamide, (19)F-FB-NAPamide, was determined to be 7.2 +/- 1.2 nM (mean +/- SD) using B16/F10 cells and (125)I-(Tyr(2))-[Nle(4),D-Phe(7)]-alpha-MSH [(125)I-(Tyr(2))-NDP] as a radioligand. The biodistribution of (18)F-FB-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16/F10 melanoma tumors with high expression of MC1Rs and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Biodistribution experiments showed that tumor uptake values (percentage injected dose per gram of tumor [%ID/g]) of (18)F-FB-NAPamide were 1.19 +/- 0.11 %ID/g and 0.46 +/- 0.11 %ID/g, in B16/F10 and A375M xenografted melanoma at 1 h after injection, respectively. Furthermore, the B16/F10 tumor uptake was significantly inhibited by coinjection with excess alpha-MSH peptide (P < 0.05), indicating that (18)F-FB-NAPamide specifically recognizes the MC1R in living mice. Small-animal PET of (18)F-FB-NAPamide in mice bearing B16/F10 and A375M tumors at 1 h after tail vein injection revealed good B16/F10 tumor-to-background contrast and low A375M tumor-to-background ratios. CONCLUSION: (18)F-FB-NAPamide is a promising molecular probe for alpha-MSH receptor-positive melanoma PET and warrants further study.